Lemon and Lime Citrus Essential Oils - ACS Symposium Series (ACS

Chapter 8

Lemon and Lime Citrus Essential Oils Analysis and Organoleptic Evaluation

Downloaded by UNIV OF MICHIGAN ANN ARBOR on February 18, 2015 | http://pubs.acs.org Publication Date: April 6, 1993 | doi: 10.1021/bk-1993-0525.ch008

Theresa S. Chamblee and Benjamin C. Clark, Jr. Corporate Research and Development Department, Coca-Cola Company, P.O. Drawer 1734, Atlanta, GA 30301

The techniques used in this laboratory to obtain accurate qualitative and quantitative analyses of lemon and lime essential oils are reviewed This quantitative database has helped to clarify the differences among Sicilian and California lemon peel oils, Mexican lime peel oil, and also to explain the chemical changes that take place during production of distilled lime oil. The HPLC-separated oxygenated fractions of Sicilian lemon peel oil have been evaluated organoleptically by a panel of experts using a capillary G C sniff port. A relatively quick, convenient method was used to pinpoint important contributors to lemon aroma. Although the data is based on a small number of opinions, the results agree well with earlier literature and expand the knowledge of lemon flavor. Relative intensity factors were assigned to lemon oil oxygenated constituents which were then ranked according to their importance to lemon aroma. Many unidentified trace constituents were also evaluated and ranked.

Lemon and lime essential oils, used principally as flavors and fragrances, are of considerable commercial importance. The U.S. is a leader in lemon oil production, accounting for about 25% of the world production in the 1980's (7). In addition, large quantities of lime and lemon oil are imported into the United States making them second and third,respectively,in terms of import dollars spent on essential oils (2). Considering the economic importance of these oils, there is a need to study them in detail. Three areas of information are required in order to understand the complex nature of essential oils: qualitative (which constituents are present), quantitative (how much of each constituent is present), and organoleptic (which constituents are most important to the flavor and end use of the essential oil). The latter can be the most important but it is often the least understood. In order to better evaluate an essential oil and its usefulness, all three areas should be explored. This paper will review some of the techniques that have been helpful to us in developing a qualitative and 0097-6156/93/0525-0088$06.00/0 © 1993 American Chemical Society

In Bioactive Volatile Compounds from Plants; Teranishi, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.

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quantitative database for lemon and lime oils, and it will also present some details of an approach used to evaluate a lemon oil organoleptically.


Qualitative Analysis: L C and GC-MS The most widely used methods for separation, component identification, and quantitative analysis of essential oils are GC and GC-MS. As is well known, even capillary GC and GC-MS are inadequate to deal completely with the many coeluting and partiallyresolvedcomponents found in these samples. Normal phase LC, both open column and especially HPLC, are useful prefractionation tools that can greatly facilitate the GC-MS identification of essential oils. By combining LC and GC, one is able to take advantage of multiple separation mechanisms. Normal phase L C separates on the basis of polarity and affinity of the compounds for silica while GC adds the effects of vapor pressure and temperature. This combinationresultsin very powerful separations. In the early 1980's we developed an HPLC method for the prefractionation of citrus essential oils and terpene mixtures (5-5). The essential oils are initially separated by open column chromatography into two fractions, first by eluting with hexane toremovethe hydrocarbons, and then with CHJC^ toremovethe oxygenated constituents. Each fraction is then further separated by normal phase silica HPLC into several sub-fractions that can be analyzed by GC-MS or other techniques. One last fraction (OC-3) was obtained by washing the open silica gel column with a 10:90 mixture of methanol-methylene chloride after most of the oxygenatedfractionwas removed with CHjClj. The HPLC system that is used consists of a Waters M6000 pump with 3 columns connected in tandem: Whatman Partisil 5 μπι, 10 μπι, and a Waters silica radial compression column. Refractive index detection is used. One advantage of normal phase liquid chromatography is the use of a nonaqueous mobile phase which allows for very easy sample work-up with no extractions necessary before further analysis of the collected samples. A series of mobile phases was tried with the best overall separations being achieved with a ternary solvent mixture of 8% ethyl acetate in a 50:50 mixture of hexane/methylene chloride. Normal phase HPLC provides a good class separation of the citrus essential oils. Elution from the silica column occurs in order of increasing polarity. The monoterpene hydrocarbons elute first, followed by the sesquiterpenes, esters, aldehydes, and finally alcohols. Figure 1 shows an HPLC of the oxygenated fraction of Sicilian lemon oil. The largest peak (fractions 6 and 7) in the chromatogram is principally citral which comprises close to 60% of the total oxygenatedfraction.By fractionating this sample using HPLC, it was possible to greatly simplify the chromatograms for GC-MS, as shown in Figure 2. The chromatogram in Figure 2A shows the Sicilian lemon oil oxygenatedfraction,and GC's of two representative sub-fractions (#8 and #12) collected from the HPLC are shown in 2B and 2C respectively. It is easy to see that GC-MS of thesefractionswill be simpler than that of the starting material. Also there is the added advantage that certain compounds which coelute in GC are separated by HPLC.



90

ι

0

BIOACTIVE VOLATILE COMPOUNDS FROM PLANTS

—

5

1

1

1

1

10

15

20

25

Minutes

Figure 1. HPLC of Sicilian lemon oil, oxygenated fraction, with a few representative compounds listed: fractions 1 and 2, solvent and trace monoterpene oxygenates; fraction 3, neryl acetate, nonanal; fraction 4, geranyl acetate, octanal; fraction 5, neral; fraction 6, neral, geranial; fraction 7, geranial; fraction 8, terpinen-4-ol; fraction 9, linalool; fraction 10, campherenol; fraction 11, unidentified;fraction12, r-sabinene hydrate, borneol;fraction13, citroncllol; fraction 14, α-terpineol; fraction 15, geraniol.


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Lemon and Lime Citrus Essential OUs


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Figure 2. Capillary GC separation employing a DB-5 column: (A) starting material, Sicilian lemon oil oxygenates; (B) HPLC fraction #8 (see Figure 1); (C) HPLC fraction #12.


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B I O A C n V E VOLATILE COMPOUNDS F R O M PLANTS

The hydrocarbon constituents are also well separated by normal phase HPLC (6). The optimum mobile phase in this case is 100% hexane. Monocyclic and bicyclic hydrocarbons arereasonablywell separated and the sesquiterpene hydrocarbons are wellresolved.This was especially useful when identifying a flavor impact compound of expressed lime oil, germacrene B. The final identification of this compound was made possible with a sample isolated and purified by HPLC (6).


Quantitative Analysis: Lemon and Lime The identification of additional constituents in lemon and lime oils has allowed us to improve and expand our quantitative database for these essential oils. A good quantitative database, obtained usingresponsefactors and internal standards, is useful for many applications. Unfortunately, as Shaw has pointed out (7), much of the analytical data available in the literature for citrus oils is based onrelativepeak area percents without the use ofresponsefactors and internal standards. Our quantitative work has provided absolute weight percent values for 52 constituents in Sicilian and California lemon oils (8), 50 constituents in Mexican expressed lime oil, and 42 constituents in Mexican distilled lime oil (9). These analysesrepresentthe most complete published quantification of these oils at present A summary by compound class of quantitative data for lemon and lime oils is shown in Table I. A complete compilation of the quantitative data for individual constituents is available (8, 9). Table I. Quantitative Volatiles Analysis: Summary by Compound Class Absolute weight percent Sicilian Lemon*

California Lemon''

Expressed Lime

Distilled Lime

Hydrocarbons, C(10) Bicyclic Monocyclic Total

14.62 76.44 91.06

15.17 76.32 91.49

25.36 53.59 78.95

3.31 72.13 75.44

Hydrocarbons, C(15)

1.16

1.24

5.70

3.48

Aldehydes

3.78

2.32

5.00

0.48

Alcohols

0.61

0.61

1.05

13.41

Esters

0.96

1.18

0.44

0.15

Ketones, Ethers

0.03

0.03

0.04

3.96

Unidentified

0.20

0.29

0.76

2.38

97.80

97.16

91.94

99.30

5

Total GC Volatiles

c

c

Corrected for GC flame detectorresponseand using tetradecane as internal standard. Fromref.8. Fromrcf.9. b

c



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Quantitative data have proved very useful for explaining differences among the lemon and lime oils. For example, the data in Table I show that the Sicilian lemon oil is rich in aldehydes but slightly lower in esters compared to the California lemon oil. Expressed lime oil, which is obtained directly from the peel of the fruit, has arelativelyhigh level of aldehydes, especially citral, and high levels of bicyclic hydrocarbons compared to lemon oil. In addition, expressed lime oil also contains significant amounts of nonvolatile material. Differences between the expressed and distilled lime oils are also evident. Distilled lime oil is rich in alcohols and low in aldehydes and bicyclic hydrocarbons compared to the expressed lime oil. The quantitative data can be used to understand the changes that take place during the production of distilled lime oil, which is actually areactionflavor made by acidcatalyzedreactionof crushed lime fruit The acid-catalyzed reactions of the bicyclic hydrocarbons that occur during production of distilled lime oil result in an increase of terpene alcohols, especially α-terpineol. The terpene aldehydes are also almost completely lost during production. Thesereactionshave been summarized in detail (9). Organoleptic Analysis In general, the more extensive the identification of constituents in these oils, the more accurate the quantitative analysis can be. Quantitative data provide a basis for understanding the correlation between quality and composition. The third component required for this correlation is organoleptic analysis. Organoleptic analysis is very complex and encompasses many variables that are hard to control, yet it has received much attention over the years. This is not an especially standardized area of study and many methods have been tried. One well known approach focuses on the use of odor thresholds which are used to calculate an aroma value or odor unit (10,11). Odor units are ratios of the concentration of a particular constituent in a food or flavor to the calculated threshold of the component They have been used extensively by Guadagni, Buttery, Teranishi and coworkers (72). Threshold values have been calculated for many individual standards in water. Pure standards of the compounds of interest are necessary and sometimes difficult to obtain. Nevertheless, this technique has been used to identify important flavor constituents in many vegetables and fruits. Sugisawa and coworkers have used a similar approach to study odor quality of orange (13). Another interesting flavor evaluation was performed by Casimir and Whitfield on passion fruit using flavor impact values (FTV) which were correlated to taste panel scores (14). Other approaches for organoleptic evaluation use a capillary sniff port to help calculate thresholds. For example, Drawert and Christoph used aroma values to rank the importance of 13 constituents in the headspaces of lemon oil, lemon peel, and lemon oil emulsion (75, 76). These aroma valuesrepresentthe ratio of the headspace concentrations of the constituent to the thresholds determined using a sniff port Grosch and coworkers also investigated lemon with a GC sniff port method using flavor dilution values (77) for threshold calculation. Flavor dilution values were calculated for twelve constituents in fresh lemon oil (18). The flavor dilution technique has also been applied to aged and oxidized lemon oil samples (79) and



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many other flavors. Acree's "charm" analysis is a similar capillary sniff port method. It involves calculation of charm values which arerelatedto thresholds and odor units (20). The method involves the capillary sniff port analysis of a series of dilutions of the sample of interest and can be applied to components of unknown identity or concentration (21, 22). Threshold information is very useful in flavor analysis; however, the approach does have some limitations. Threshold determination is time consuming and generally requires a large, trained panel. To be most effective, thresholds should be determined in a medium that is appropriate for the sample of interest (23,24). There is often large variability in threshold measurements reported from laboratory to laboratory (25). This may be due to the fact that pure standards are often difficult to obtain. In the case of unidentified constituents and even some known compounds, pure standards are not available. The capillary sniff port methods avoid some of these limitations. Unknown constituents can be evaluated; but, the possibility of coeluting trace constituents still exists. Analyzing several dilutions of a complex essential oil by capillary sniff port can be very time consuming. Another issuerelatingto thresholds is the possibility of the additive effects of sub-threshold constituents in a mixture. There are several reports in the literature describing the additivity of flavor compounds to produce an aroma even when they are present below their individual thresholds (24, 26-30). Thesereportsgive strong evidence that compounds present in a mixture below their threshold should not be ignored. Capillary Sniff Port Approach for Lemon Oxygenates The above issues and limitations have led us to a somewhat simpler, quicker approach for organoleptic analysis that uses a capillary GC sniff port and does not rely on threshold determination. A similar method for generation of initial intensity data was used by von Sydow et al. to study bilberry flavor (31). The objective of our analysis was to determine the relative importance of constituents, particularly trace unknowns, present in the oxygenated fraction of Sicilian lemon oil. Efforts were concentrated on the oxygenates because a preliminary sniff port test of the hydrocarbon fraction suggested that it is much less important to the lemon aroma. Citral is a well known lemon flavor impact compound, but it would be useful to determine the significance of the other constituents as well. Over 140 compounds have been reported to be present in lemon peel oil (32). Unfortunately, some of these identifications werereportedwith little or no supporting analytical data. We have confirmed the presence of over 60 compounds in Sicilian lemon oil (8 and (Clark, Jr., B. C.; Chamblee, T. S.; The Coca-Cola Company, unpublished data)). Our GC-MS analysis of the HPLC fractions for the lemon oil oxygenates indicated «150 compounds that were not identified or confirmed by us. Most of these are trace constituents because only approximately 0.20 weight % of lemon oxygenates remain unidentified (8). Trace constituents have been found to be important in other essential oils (33). Since identification of trace constituents can be difficult, it would be especially helpful to know which of the unidentified compounds are organoleptically important This capillary sniff port approach does not attempt to


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provide arigorousorganoleptic evaluation of the oil but rather is designed to be used as a guideline for pinpointing important aroma constituents. Experimental. A panel of five people, chosen especially for their familiarity with citrus flavors, was used. The panelists knew they were smelling lemon fractions and were instructed to note any odors that were especially lemony or citrus-like. However, during the study they were not aware of which fraction they were smelling at a particular time or which compounds might be present in the fraction. In two sessions, the lemon fractions were first evaluated by blotter sniff test. The panelists smelled 15 fractions collected from the HPLC (Figure 1) and one fraction (OC-3) of additional polar constituents eluted from the open silica gel column with methanolmethylene chloride (10:90, v/v) after the methylene chloride fraction. A GC of the open column fraction showed that it contained trace amounts of most of the oxygenated constituents. The panelists were asked to describe each fraction with respect to its importance to lemon flavor. Thefractionscontaining most of the citral (fractions #6 and 7) were described as very lemon-like, as expected. However, interesting comments, including additional citrus notes wererecordedfor several of the other fractions as well. These results were used to choose six fractions for further analysis by capillary sniff port: #2, 3, 4, 11, 12, and OC-3. The capillary sniff port set-up is installed in a Varian 3700 GC and it has the advantage of being very simple. An SGE fused silica capillary outlet splitter is attached at the end of the capillary column. The splitter consists of two capillaries of different diameter yielding a splitratioof 10:1. The capillaries of the splitter are connected so that ten parts of the GC effluent go to the sniff port and one part goes to the FID detector. The sniff port, which is available from Varian, consists of a heated tube inside an empty thermal conductivity detector block through which the larger capillary is threaded to the atmosphere. The detector block has an independent temperature control set at 240°C. A stream of humidified air has been added at the sniff port outlet to prevent nose dry-out, although this does not seem to be much of a problem with this set-up, probably because the panelist is only smelling the flow coming from the capillary column, «1-5 ml/min. A megaborc (0.5mm i.d.) DB-5 capillary column was used. There was an excellent correlation ofretentiontime between the FID and sniff port outlet, no band broadening, and no carry over from peak to peak even with very large and intense peaks. The typical sample concentration injected was generally in the ng/peak or smaller range. However, it is well known that peak size is not necessarily indicative of odor intensity. Prefractionation of essential oils isrequiredfor sniffing because a whole oil chromatogram will have many overlapping peaks. Also, the panelist can be overwhelmed by too many odors if the column is overloaded. An attempt was made to inject just enough sample on the GC so that the large peaks would not be too overpowering. Therefore, in arelativelydilute run a strong odor would denote a fairly potent compound, especially if the peak was very small. The sniffers were instructed to smell the GC effluent, describe the aroma in general flavor terms, and make an estimate of the intensity for each odor detected. The panelists did not pay particular attention to whether a peak was eluting or not A second personrecordedaroma descriptions and intensity evaluations directly on



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the chromatogram at the retention time where they were noted. Each session lasted »60 minutes. Figure 3 shows a sample GC sniff port chromatogram. Intensity evaluations were converted to a numerical scale: 1= very weak, 2 = weak, 3 = moderate, 4 = strong, 5 = very strong. The HPLC exhibits tailing of some of the larger components (Figure 1),resultingin some carry over of major constituents from fraction to fraction. Therefore, several constituents are present in more than one fraction, especially citral which comprises «60% of the total oxygenated fraction. This turned out to be an advantage for sniff port analysis because it allowed for smelling of many of the constituents at different concentrations without going through dilutions of the sample. Results. Correlation ofrelativeretentiontime data with previous GC-MSresults(8) allowed for identification of the known major constituents of lemon in the fractions that were analyzed by sniff port. A few identifications remain tentative(t). An analysis of the comments showed that the following identified peaks were described as citrus or lemon-like by more than one panelist: citronellal, citronellol, decanal, dodecanal, geranial, geraniol, linalool, neral, nonanal, octanal, perilla aldehyde(t),

Lemon and Lime Citrus Essential Oils - ACS Symposium Series (ACS

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